The following protocol presents how to set up a Neutron Spin Echo experiment to measure the intermediate scattering function and investigate the dynamics of proteins in solution. The main advantage of Neutron Spin Echo Spectroscopy is its ability to look at rearrangement of protein domains and sub domains on the pico to nanosecond time scale;which is the range of slow motions in proteins in their nearly natural environment, and in the crowded protein solution. Neutron Spin Echo is also sensitive to isotopic configuration, which allows very specific and targeted studies using contrast matching.
Insights into protein domain dynamics is a major part of biophysical research in the ongoing difficult task to relay the proteins'domain motions with their biological functionality. The protocol presented here can be generally applied to any Neutron Spin Echo measurement performed at the SNS-NSE spectrometer, regardless of your choice of samples, if one stays within the realm of soft matter materials. To set up the experiment begin by selecting the thickness of the cell sample loading, based on the concentration of the protein sample, the temperature needed for measurement, and the amount of solution available.
Clean the cell with phosphate-free dish detergent, deionized water, and 70%diethanol. Dry the cell in the convection oven. Load four milliliters of protein solution into the cell, and close with caps.
Use wax film or any sealant to seal the sample cells. Load four milliliters of dialysis buffer into an identical container as the protein sample and seal. Transport samples to the beam line.
Close the shutter, and enter the spectrometer enclosure cave area. Mount the sample cell on the aluminum sample holder by tightening the screws and the holding plates. Mount the graphite sample and aluminum oxide powder sample loaded in an identical container as the protein sample.
Place the same holder by gently sliding it into the can of the temperature forcing system. Close the temperature forcing system lid, and set the temperature to the desired value by accessing the interactive screen of the temperature forcing system. Mount the neutron camera for the alignment of the samples to the beam.
To collect the Neutron Spin Echo Spectroscopy data, align the sample in the neutron beam using the neutron camera, and the four independent sample apertures. Open the Spallation Neutron Source, Neutron Spin Echo Spectroscopy data collection software, and collect sample statistics by running defraction scans for the desired scattering angles and wavelength. Set up the measurement parameters based on the statistics collected for each sample by editing the measurement macros provided by the Assisting Instrument Scientist.
Start scanning by typing the protocol name at the command prompter and acquiring echoes for the sample. Log in to Neutron Sciences Remote Analysis cluster with the ORNL user credentials and press the launch session button. In the user directory, open a terminal window and type the software setup command.
Next, type the environment command. Create a folder for the data reduction in the home directory and copy the provided scripts and macros from the shared directory. Edit, rename and save the reduction macro provided accordingly.
Type drspine"at the command prompter and press Enter to start the software reduction environment. Type the name of the reduction macro in the command prompter within the software environment, and press Enter. Edit the Python script provided by the Assisting Instrument Scientist with the names of the reduced file data.
Edit the function to fit from the library provided. Type the name of the edited script at the command prompter, and press Enter to read, fit, and plot reduced NSE data. The intermediate scattering function measured by NSE for IgG and MBP proteins shows a clear deviation from a simple diffusion-like relaxation process on the short four year time scale, indicating the accessibility of protein internal dynamics by NSE, and the need for a more complex model to describe the observed dynamical processes.
The results of intermediate scattering function calculations fitted by atomic modeling of both proteins using models developed by were in excellent agreement with the experimental NSE data. The results proved that slower dynamics observed at longer furrier times can be attributed to the overall translational and rotational diffusion processes, while the dynamics observed at the short time scales can be attributed to the dynamics of protein domains. One important thing to remember is the need of well prepared and clean samples for NSE measurements.
A good sample for a Spin Echo has a flip ratio higher than three, which is the ratio between coherent and incoherent scattering of the sample. Also during sample preparation and sample loading into the NSE cells, the sample solution should be kept safe and free from any chemical or biological cross contamination. For safety reasons the entrance in the spectrometer enclosure should only be attempted after the shutter is closed, and an additional half an hour time has been allowed for radiation cooling of the enclosure.
Small-Angle Neutron Scattering is recommended to assess the shape and the structure of proteins in a concentrated solution. It can be done before or in parallel with the NSE experiment. Additional methods like dynamic light scattering and viscosity measurements provide information about the translational diffusion and the hydrodynamics interaction within the concentrated protein solution, and are also recommended.
